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MINIREVIEW
The undecided serpin
The ins and outs of plasminogen activator inhibitor type 2
Robert L. Medcalf and Stan J. Stasinopoulos
Australian Centre for Blood Diseases, Monash University, Prahran, Victoria, Australia
Introduction
The plasminogen activating cascade became a much
investigated enzyme system during the early 1980s,
mainly for its role in maintaining vascular patency and
for its effect on the extracellular matrix in the context
of wound healing and cell migration. The controlled
generation of the powerful protease, plasmin, from
its precursor plasminogen seemed to be a relatively
straightforward process at the outset: two serine pro-
teases had been identified that could specifically cleave
plasminogen and produce active plasmin. These pro-
teases (tissue-type- and urokinase-type plasminogen
activator; tPA, uPA) were in turn specifically inhibited
by plasminogen activator inhibitors (PAIs)-types 1 and
2, both of which belong to the serine protease inhibitor
(serpin) superfamily. Other cofactors, such as the ser-
pin alpha
2
antiplasmin, the urokinase receptor (uPAR)
and fibrin, were also shown to play important roles in
regulating plasmin formation and activity [1]. This
may have been the general consensus in the late 1980s,
but nowadays it has become clear that many of the
individual components of the fibrinolytic ⁄ plasminogen
activating system perform other roles that could not
have been foreseen. tPA, for example, is not just a

‘plasminogen activator’; it is now widely appreciated
for its role in the central nervous system [2,3].
Although it can act on its classical substrate, plasmino-
gen, in this compartment, it also associates with other
targets, and in some cases can even act like a cytokine
to activate microglial cells without engaging its cata-
lytic properties [4]. Similarly, the two plasminogen acti-
vator inhibitors are now known to perform additional
functions. PAI-1 can act as an accessory protein that
modulates the association of the uPA receptor with in-
tegrins. This association, in turn, influences cell migra-
tion independently of the PAI-1 protease inhibitory
activity [5,6].
Keywords
gene regulation; plasminogen activator
inhibitor type 2; protease inhibitor; serpin
Correspondence
R. L. Medcalf, Australian Centre for Blood
Diseases, Monash University, 6th Floor
Burnet Building, 89 Commercial Road,
Prahran, 3181 Victoria, Australia
Fax: +61 39903 0228
Tel: +61 39903 0133
E-mail:
(Received 31 March 2005, accepted 13 July
2005)
doi:10.1111/j.1742-4658.2005.04879.x
Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine
protease inhibitor (serpin) with unique and tantalizing properties that is
generally considered to be an authentic and physiological inhibitor of uro-

kinase. However, the fact that only a small percentage of PAI-2 is secreted
has been a long-standing argument for alternative roles for this serpin.
Indeed, PAI-2 has been shown to have a number of intracellular roles: it
can alter gene expression, influence the rate of cell proliferation and differ-
entiation, and inhibit apoptosis in a manner independent of urokinase inhi-
bition. Despite these recent advances in defining the intracellular function
of PAI-2, it still remains one of the most mysterious and enigmatic mem-
bers of the serpin superfamily.
Abbreviations
ARE, AU-rich element; IL, interleukin; K5, keratin 5; LPS, lipopolysaccharide; ov, ovalbumin; PAI, plasminogen activator inhibitor; PAUSE-1,
PAI-2-upstream silencer element-1; Rb, retinoblastoma; serpin, serine protease inhibitor; TNF, tumour necrosis factor; tPA, tissue-type
plasminogen activator; TTP, tristetraprolin; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor.
4858 FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS
For PAI-2, there was a strong suspicion soon after
its discovery that the real function of this inhibitor
had been overlooked. From a teleological viewpoint,
a non-uPA inhibitory role was expected, as the
majority of PAI-2 was found in a location where its
intended or perhaps presumed natural target (i.e.
uPA) did not even exist, that being the cell cytosol
[7]. This minireview will focus on the cellular and
molecular biology of PAI-2 and highlight some of
the most recent findings on the role and impressive
pattern of regulation of this enigmatic protease inhi-
bitor. Although new data is emerging, PAI-2 is still
one of the most cryptic protease inhibitors known
and its role in biology and pathophysiology is still
being unravelled.
General biology of PAI-2
PAI-2 was defined as a placental tissue-derived uPA

inhibitor over three decades ago [8], which was subse-
quently verified by others [9,10]. Human PAI-2 con-
sists of a single chain protein of 415 amino acids
encoded by a 1900 bp PAI-2 transcript and is highly
homologous with mouse and rat PAI-2. PAI-2 exists
predominantly as a 47 kDa nonglycosylated intracellu-
lar form [7], however a small percentage of PAI-2
is able to enter the secretory pathway by a process
referred to as facultative translocation [11] and secre-
ted as a 60 kDa glycosylated protein. The basis for this
bi-topological distribution is due to the lack of a con-
ventional hydrophobic amino-terminal signal sequence.
Instead, PAI-2 possesses an inefficient internal signal
sequence [12]. This bi-topological (intracellular ⁄ extra-
cellular) expression pattern of PAI-2 has also been
shown for a related serpin known as maspin [13] and
this feature remains one of the most intriguing aspects
of PAI-2 (and maspin) biology. Because the uPA
inhibitory capacity of both forms of PAI-2 seems to be
similar, it was considered early on that the release of
high local concentrations of nonglycosylated PAI-2
from dead or dying cells at sites of inflammation may
provide an immediate source of enriched uPA inhibi-
tory activity [14]. While anecdotal evidence would cer-
tainly support this, the growing consensus of opinion,
however, is that PAI-2 possesses an as yet ill-defined
intracellular role.
Structural considerations
Based on a number of criteria, PAI-2 has been
classed as a member of the ovalbumin subfamily of

serine protease inhibitors known as the ovalbumin
(ov)-serpins, with ovalbumin being the prototypical
member of this family [15]. Ovalbumin-serpins share
a common genomic structure and all lack conven-
tional signal sequences and are, for the most part,
located intracellularly. Closer examination of the
genomic structure of PAI-2 revealed another distinctive
feature, that being an extension of exon 3 that encoded
a unique domain bridging helices C and D of the pro-
tein. This so-called C-D interhelical domain [16], other-
wise known as the C-D loop, has since been implicated
in the function of PAI-2. Glutamine residues in the
C-D loop can be crosslinked by tissue transglutaminase
and factor XIII to structures in trophoblasts and to
fibrin [16–18]. Moreover, the C-D loop has been
shown to bind noncovalently to annexins, retinoblastoma
protein and a number of unidentified proteins [19,20].
Using the expressed C-D interhelical loop as bait, Fan
et al. identified the b1 subunit of the proteosome as
a binding partner [21]. The physiological relevance
of these findings remains to be clarified, but none-
theless points to diverse roles of the C-D loop in
PAI-2 function.
Polymerization of PAI-2
Many serpins have been shown to undergo loop sheet
polymerization. Generally, polymerization occurs due
to a genetic aberration, which results in serious patho-
logical consequences due to conformational changes of
these proteins [22]. PAI-2 is also able to polymerize,
but in contrast to the other polymerizing serpins this is

not a consequence of a mutation in the PAI-2 gene,
nor is it associated with any known pathologies.
Indeed, PAI-2 displays conformational plasticity and is
the only known wild type serpin to form polymers
spontaneously and reversibly under physiological con-
ditions [23]. Furthermore, this is influenced by the
redox status of the cell: PAI-2 can exist in either a sta-
ble monomeric or a polymerogenic configuration, the
latter stabilized by disulfide bonds that connect a cys-
teine residue within the C-D loop to another cysteine
residue at the bottom of the molecule [24]. The mono-
meric form is also stabilized by binding to vitronectin
while retaining its inhibitory activity. Under conditions
of oxidative stress, the polymerized inactive configur-
ation of PAI-2 can form but whether this has any
other impact on cell function is unknown. More
recently it was shown that the C-D loop within the sta-
ble monomeric form of PAI-2 is mobile and that the
monomeric and polymerogenic forms of PAI-2 were
interchangeable [25]. Hence, not only does PAI-2 exist
as a bi-topological protein, it can also exist in different
conformational forms within the intracellular compart-
ment.
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FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS 4859
Expression pattern of PAI-2 and its role
in pregnancy
Under normal conditions, PAI-2 has a restricted tissue
distribution pattern with expression detected at high
levels in keratinocytes, activated monocytes and the

placenta [26]. Lower constitutive levels of PAI-2 are
also found in other cells, including cells of neuronal
origin [27]. Plasma levels of PAI-2 are usually low or
undetectable; however, they rise significantly in some
forms of monocytic leukaemia [28]. One of the most
physiologically striking observations for PAI-2 con-
cerns its association with pregnancy. Plasma levels of
PAI-2 increase impressively during the third trimester
of pregnancy (up to 250 ngÆmL
)1
) and are maintained
at these levels for up to 1 week postpartum and then
rapidly decline [10]. The tissue source of plasma PAI-2
is the placenta itself. Indeed, PAI-2 is highly expressed
in trophoblasts [29,30] and it was conjectured that
PAI-2 acted to protect the placenta from proteolytic
degradation towards the end of the gestational period
and to regulate postpartum haemostasis. However, a
placental associated PAI-2 sensitive protease is yet to
be described. Perhaps the role of PAI-2 in the placenta
is unrelated to protease inhibition. In this regard, it is
interesting to point out that PAI-2 forms complexes
with other placental proteins, including vitronectin
[9,31], but the functional significance of this in terms
of placental biology is unknown.
The association of PAI-2 with pregnancy and its
placenta-specific expression suggested that PAI-2
might perform a critical function during foetal devel-
opment. If this were indeed the case, one would have
predicted a developmental abnormality in PAI-2

– ⁄ –
mice. Mice with a targeted deletion in the PAI-2 gene
have been described but these mice have not as yet
displayed any noticeable phenotype [32] at least under
normal, nonchallenging conditions. To exclude the
possibility that the lack of effect was due to redund-
ancy with PAI-1, a double knock-out mouse was pro-
duced that harboured a disruption at both the PAI-1
and PAI-2 loci. Still, no obvious phenotype was seen.
Given the high degree of PAI-2 expression in the
human placenta, it was surprising at first glance that
foetal development and reproduction was undisturbed
in PAI-2
– ⁄ –
mice. However, no firm conclusions can
be drawn from this as, unlike the human situation,
PAI-2 is not found in the mouse placenta. It is
indeed a strange curiosity that the presence and regu-
lation of placental PAI-2 is not conserved in the
mouse. However, this important data has only been
presented as a statement within a review article [33]
and additional supportive information would be
welcomed on the presence or absence of PAI-2 in the
mouse placenta.
The role of PAI-2 in the skin
PAI-2 expression within the skin is restricted to the
upper layers of the dermis. PAI-2 has also been repor-
ted to inhibit keratinocyte proliferation [34] and to
play a role in keratinocyte differentiation [34]. A
cleaved form of PAI-2 has been found in keratinocytes

[35] implying that PAI-2 itself is a substrate for a pro-
tease in these cells.
To determine the consequences of dysregulation of
PAI-2 on epidermal differentiation, Zhou et al. [36]
produced transgenic mice that overexpressed PAI-2 in
the proliferating layers of mouse epidermis and hair
follicle cells by placing the PAI-2 transgene under the
control of the keratin 5 (K5) promoter. Although the
presence of PAI-2 had no effect on skin morphology
or proliferation under normal conditions, the PAI-2
overexpressing mice were found to be highly suscept-
ible to chemically induced papilloma formation. The
means by which PAI-2 promoted papilloma formation
is unknown, but may have been related to its reported
antiapoptotic effect (see below) since cessation of
tumour promoting treatment in control mice resulted
in extensive apoptosis of the papilloma but not in the
K5-PAI-2 transgenic mouse.
Leukocyte biology
Monocytes and macrophages express PAI-2 and levels
are impressively increased in these cells following sti-
mulation with tumour necrosis factor (TNF) [14] and
lipopolysaccharide (LPS) [37,38]. Induction of PAI-2
gene expression has been associated with monocyte dif-
ferentiation, at least in the U-937 monocyte-like cell
system [39], suggesting a role for PAI-2 in this process.
In the mouse system PAI-2 does not appear to be
indispensable for leukocyte development as PAI-2
– ⁄ –
mice exhibit normal leukocyte recruitment and appear

to differentiate normally [32].
Novel insights into the role of PAI-2 in monocytes
came from studies using THP-1 cells. Unlike all other
widely used monocyte-like cell lines (e.g. U-937, K562,
HL60) that express endogenous PAI-2, the THP-1
monocytic cells provided a notable exception to this
rule. THP-1 cells bear many features common to regu-
lar mononuclear phagocytes, but are closer in pheno-
type to a mature monocyte than other monocytic cell
lines (i.e. U-937, K562). Although the expression pat-
tern of THP-1-derived uPA and its receptor (uPAR) is
similar to that observed in other monocytic cell lines
The undecided serpin R. L. Medcalf and S. J. Stasinopoulos
4860 FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS
[40,41], THP-1 cells do not express a functional PAI-2
protein [40]. These authors demonstrated that THP-1-
derived PAI-2 was functionally inactive while the PAI-2
transcript in these cells was truncated.
The molecular basis for the aberrant production of
PAI-2 in THP-1 cells is due to a translocation anomaly
[42]. The complete absence of a functional PAI-2 in
these cells defined THP-1 cells effectively as a human
monocytic PAI-2
– ⁄ –
cell line. To take advantage of this
PAI-2
– ⁄ –
cell line, Yu et al. [43] produced stable THP-1
cell lines that expressed either wild type PAI-2 or a
PAI-2 mutant containing an alanine substitution at the

P1 position (Arg380). The presence of wild type PAI-2
caused a significant decrease in THP-1 cell prolifer-
ation, reduction in DNA synthesis and a phenotypic
change following phorbol ester-induced differentiation.
The ability of PAI-2 to alter the differentiation process
was dependent on its active form as cells expressing
PAI-2
Ala380
did not display these changes. This study
demonstrated for the first time an intracellular role for
active PAI-2 in monocytes. These results were con-
sistent with the possibility that PAI-2 disrupted an
intracellular protease(s) that was involved in cell prolif-
eration and ⁄ or differentiation although no such target
protease has been detected thus far.
PAI-2 is also present at very high levels in eosino-
philic leukocytes. Indeed the level of PAI-2 in these
cells was shown to be the highest among all other leu-
kocyte subtypes [44]. Furthermore, PAI-2 was localized
to eosinophil-specific granules and shown to be still
capable of inhibiting urokinase. It was suggested that
PAI-2 might play a role in eosinophil mediated inflam-
mation and tissue remodelling.
Role of intranuclear PAI-2
A number of Ov-serpins have been detected within the
nuclear compartment, including bomapin, PI-9, and
maspin [45–47]. PAI-2 has also been shown to have a
nuclear presence [20,45,46] yet the physiological role of
PAI-2 in this compartment is unknown. However, in
a study by Darnell et al. nuclear-located PAI-2 was

shown to bind to retinoblastoma protein (Rb) via its
CD-loop [20]. Rb is a prototypical tumour suppressor
gene and critical cell cycle regulator that targets the
E2F family of transcription factors [48]. PAI-2 colocal-
ized with Rb and, interestingly, inhibited Rb turnover
by protecting Rb from proteolysis [20]. This in turn
led to an increase in Rb protein levels and Rb-medi-
ated activities including the transcriptional repression
of oncogenes. This is a curious finding because PAI-2
– ⁄ –
mice do not appear to have any change in cell number,
and it would be predicted that Rb turnover would be
accelerated in PAI-2
– ⁄ –
mice freeing E2Fs to mediate
proliferation. Although additional evidence is required
to explore the consequences of PAI-2 and Rb inter-
action, these data underscore a novel and previously
unsuspected intranuclear role for PAI-2.
The role of PAI-2 in metastatic cancer,
apoptosis and infection
Cancer
A number of in vivo studies have assessed the prognos-
tic relevance of tumour- and stromal-derived PAI-2 in
the metastatic spread of cancer of the neck, lung and
breast [49–53]. The only established protease target for
PAI-2, namely uPA, is strongly implicated in facilita-
ting cell dissemination in the context of tumour meta-
stasis and it is likely that the beneficial effect of PAI-2
seen in these studies is simply via uPA inhibition.

Overexpression of PAI-2 in melanoma cells prevented
spontaneous metastasis of transplanted cells in scid
mice [54], while overexpression of PAI-2 in HT-1080
cells has also been shown to reduce uPA-dependent
cell movement in vitro and metastatic development
in vivo [55]. The ability of PAI-2 to selectively bind to
cell surface bound uPA (via uPAR) and subsequently
be internalized [56] has prompted studies to assess the
effectiveness of PAI-2 as a delivery vehicle for isotopes
(
213
Bi) and toxins that can be targeted to uPA-bearing
cancer cells This approach has provided positive out-
comes at least in some preclinical studies [57–59].
Apoptosis
Circumstantial evidence that first implicated PAI-2 as
an inhibitor of apoptosis came from genetic associ-
ation studies with BCL-2 [60]. The BCL-2 proto-
oncogene was discovered over 15 years ago as the
archetype inhibitor of apoptosis. Evidence that BCL-2
was playing such a role in humans came from studies
in patients with follicular lymphoma. In these patients,
a translocation event occurs between chromosomes 14
and 18 t(14; 18) that brings the BCL-2 gene into juxta-
position with the locus of the immunoglobulin heavy
chain, resulting in overexpression of BCL-2 [61]. This
in turn inhibits the apoptotic process of the lym-
phoma. The relevance of this to PAI-2 stemmed from
the fact that the PAI-2 gene is located less than
300 mbp from the BCL-2 gene and is translocated

along with BCL-2 in patients with follicular lym-
phoma. PAI-2 and BCL-2 also share structural similar-
ities and it was proposed that the function of PAI-2
may overlap with BCL-2. So with this background, a
R. L. Medcalf and S. J. Stasinopoulos The undecided serpin
FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS 4861
number of publications in the mid-1990s provided
in vitro evidence that PAI-2 could inhibit TNF-induced
apoptosis in HT-1080 fibrosarcoma cells [62] and HeLa
cells [63]. A cleaved form of intracellular PAI-2 has
been found in ND4 monocytes undergoing apoptosis
[64]. In no case has an intracellular PAI-2-sensitive
proteinase been identified. Other reports, however,
have provided contradictory data [18]. One argument
in the interpretation of the significance of PAI-2 dur-
ing apoptosis concerns the level of enforced expression
of PAI-2 in the model systems used. In most of these
in vitro studies, PAI-2 was overexpressed in cells that
either did not make PAI-2 at all or were expressed to
levels that well exceeded endogenous expression levels.
Under these conditions, PAI-2 may indeed inactivate
one or more intracellular proteases, but whether this
genuinely reflects the in vivo role of PAI-2 can be rea-
sonably debated.
Viral infection
Evidence to suggest that PAI-2 participates in the host
response to alphaviral infection is based on over-
expression studies in HeLa cells. The protective effect
of PAI-2 was indirect, as PAI-2 appeared increase
interferon levels which then triggered an increase in

the expression of a battery of antiviral genes [65].
Shafren et al. [66] also demonstrated the same PAI-2
over-expressing HeLa cells were protected from lytic
infection by human picornaviruses. In this case, PAI-2
promoted the transcriptional down-regulation of sur-
face expression of picornavirus receptors (decay accel-
erating factor, intercellular adhesion molecule-1 and
coxsachievirus-adenovirus receptor; DAF, ICAM-1
and CAR, respectively). These observations further
support the growing body of evidence [42,43] that
intracellular expression of PAI-2 is linked to a signal-
ling pathway(s) that can reprogram gene expression.
One may even speculate that PAI-2 could play a role
in the innate immune response since its expression is
commonly associated with inflammation and the host
response to infection.
PAI-2 gene expression and regulation
Based on data accumulated over the past 17 years, it is
evident that the PAI-2 gene expression can be induced
by a wide range of agonists. Moreover the level of
PAI-2 gene induction in some examples is quite extra-
ordinary. Agonists of PAI-2 induction include growth
factors (transforming growth factor-b, epidermal
growth factor and monocyte-colony stimulating fac-
tor; TGFb, EGF and M-CSF, respectively), hormones
(retinoic acid, dexamethasone and vitamin D3), cyto-
kines [TNFa, interleukin (IL)-1 and IL-2)], vasoactive
peptides (angiotensin II), toxins (dioxin and endotoxin)
and tumour promoters (phorbol esters and okadaic
acid) [26,67]. PAI-2 mRNA expression is also strongly

increased by the excitotoxic glutamate analogue, kainic
acid in neuronal cells in vivo [27].
PAI-2 was cloned by groups that had an intent
focus on the cell and molecular biology of PAI-2
[39,68,69], and by others inadvertently through differ-
ential gene expression studies. For the latter, PAI-2
was identified as a TNF responsive gene in monocytes
and fibroblasts [70,71] and as a dioxin responsive gene
in keratinocytes [72]. Microarray studies identified
PAI-2 as an inducible gene in response to IL-5 [73],
factor 7 ⁄ tissue factor [74], and again by TNF [75]. Dif-
ferential gene expression profiling (SAGE) of LPS-trea-
ted primary human monocytes identified PAI-2 as the
third most inducible gene being induced 105-fold by
this agent [37]. In a microarray study to identify Lp(a)
inducible genes in human monocytes, PAI-2 mRNA
was found to be the most induced transcript from a
screen of 8000 cDNAs [76]. These latter studies pro-
vide further evidence of the diverse repertoire of agents
that strongly regulate PAI-2 expression and by associ-
ation, PAI-2 is likely to play a role in the biological
consequences initiated by these agents.
The impressive magnitude of induction by such a
variety of biological agents prompted many laborator-
ies to explore the transcriptional and post-transcrip-
tional processes underlying PAI-2 expression.
Transcriptional regulation of PAI-2 expression
Run-on transcription assays provided direct evidence
that the induction of PAI-2 expression in U-937 cells
following phorbol ester treatment involved dramatic

increases in the rate of PAI-2 transcription [39]. Similar
studies in HT-1080 fibrosarcoma cells demonstrated a
transcriptional component following TNF-mediated
induction of PAI-2 expression [14]. These studies led to
an analysis of the PAI-2 promoter [77,78]. DNase-1
protection experiments indicated that the proximal
region of the PAI-2 promoter possessed a congested
arrangement of cis-acting elements. Of these, only the
AP1-like elements, AP1a (TGAATCA, )103 to )97)
and AP1b (TGAGTAA, )114 to )108), and a cAMP
responsive element (CRE)-like element (TGACCTCA,
)187 to )182) [77,79] were shown to have functional
activity during transcriptional regulation. Curiously, a
repressor element located between )219 and )1100 was
suggested to play a role during TNF induction [80].
The identification of the exact sequence within this
The undecided serpin R. L. Medcalf and S. J. Stasinopoulos
4862 FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS
region and trans-acting factors responsible for this
activity have not been reported. Antalis et al. [81]
characterized 5.1 kb of 5¢ flanking region in U937 cells
by deletion analysis and found a silencer between
)1977 and )1675 that acts in an orientation- and
position-independent but not cell-specific manner. The
silencer activity was localized to a 28 bp sequence
containing a 12 bp palindrome at position )1832,
CTCTCTAGAGAG, which was termed PAI-2-
upstream silencer element-1 (PAUSE-1). Later analysis
defined the minimal functional PAUSE-1 element as
TCTN

x
AGAN
3
T
4
, where x ¼ 0, 2 or 4 [82]. UV-cross-
linking analyses determined that the PAUSE-1 binding
protein was  67 kDa, but its identity remains
unknown. In their study, PAUSE-1 was not character-
ized in the context of TNFa induction and it would
be worthwhile to explore the relationship between
PAUSE-1 and the element that selectively represses
TNFa inducibility in HT-1080 cells [80].
Post-transcriptional regulation of PAI-2 expression
As mentioned earlier, PAI-2 is one of the most highly
regulated genes known, at least in terms of the magni-
tude by which it is induced by growth factors, hor-
mones, cytokines [73,83] and tumour promoters
[39,84]. Although PAI-2 induction involves substantial
changes at the level of transcription, post-transcrip-
tional events are also important in modulating its
expression. This was first revealed over a decade ago,
when it was shown that the increase in PAI-2 mRNA
after synergetic stimulation by phorbol myristate ace-
tate and TNFa (1000- to 1500-fold) could not be
accounted for by an increase in PAI-2 transcription
rate alone (50-fold), suggesting that post-transcrip-
tional processes influence PAI-2 gene expression [84].
The PAI-2 transcript has since proven to be a valu-
able model to study post-transcriptional regulation,

most notably at the level of mRNA instability.
PAI-2 mRNA contains a functional nonameric
(UUAUUUAUU) AU-rich element (ARE) in its 3¢-un-
translated region [85]. Mutagenesis of this element par-
tially stabilized the normally unstable PAI-2 mRNA,
hence revealing a functional role for this motif [85,86].
This element also provides binding sites for several
ARE binding proteins, including the stabilizing protein
HuR [86] and the mRNA destabilizing protein tristetr-
aprolin (TTP) [87]. HuR is a member of the Hu family
of mRNA binding proteins and has been associated
with promotion of mRNA stability [88]. TTP, on the
other hand, is a potent mRNA destabilizing protein
that associates with ARE elements in cytokine tran-
scripts, including TNF a [89] and IL-3 [90]. Overexpres-
sion of TTP in HEK 293 cells transfected with a
constitutively active PAI-2 expression vector resulted
in loss of PAI-2 mRNA, suggesting that TTP can
indeed regulate PAI-2 expression [86]. Other cytoplas-
mic and nuclear proteins also bind to the ARE with
the PAI-2 3¢-UTR [85,86] but these are yet to be iden-
tified. The PAI-2 transcript also possesses another
instability determinant located within exon 4 of the
PAI-2 coding region [91]. UV-crosslinking studies have
identified two RNA-binding proteins (approximately
50–52 kDa) that specifically interact with this
sequence. Taken together, the data published to date
suggest that PAI-2 mRNA stability is influenced by
elements located within both the coding region and the
3¢-UTR. It remains to be determined whether these

instability elements in the coding region and the
3¢-UTR act in a coordinated fashion to control PAI-2
mRNA stability (Fig. 1).
Conclusion
PAI-2 has been implicated in many facets of biology
some of which are unrelated to its ability to inhibit
extracellular uPA. However, the ability of PAI-2 to
reduce the metastatic potential of a number of cancers,
Fig. 1. Schematic representation of regula-
tory domains within the PAI-2 transcript that
influence PAI-2 expression at the post-tran-
scriptional level. At least two domains exist:
one within exon 4 (E4) of the coding region
and the other within the 3¢-UTR. Proteins
that have been shown to bind to these
regions in vitro are shown. See text for
details. E, exon.
R. L. Medcalf and S. J. Stasinopoulos The undecided serpin
FEBS Journal 272 (2005) 4858–4867 ª 2005 FEBS 4863
presumably via inhibition of extracellular or cell-sur-
face bound uPA, is arguably the most consistent and
physiologically relevant finding to date. Nonetheless,
the response of the PAI-2 gene to such a diverse reper-
toire of agonists and the impressive magnitude of
induction in leukocytes in response to toxins and
cytokines invokes PAI-2, albeit circumstantially, with
inflammation, tissue repair and possibly the innate
immune response. Similarly, the evidence linking PAI-2
with apoptotic processes, Rb turnover, cell prolife-
ration and differentiation is substantial and gaining

momentum but more direct and physiologically
focused experiments are needed in order to define its
undisputed intracellular function. It is anticipated that
this information will be forthcoming through a more
extensive analysis of the PAI-2
– ⁄ –
mice. Results from
these experiments are eagerly awaited.
Acknowledgements
This study was supported by grants obtained by RLM
from the National Health and Medical Research
Council of Australia.
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